Am J Physiol Gastrointest Liver Physiol 292: G725–G733, 2007;
doi:10.1152/ajpgi.00406.2006.
Diabetes induces sex-dependent changes in neuronal nitric oxide synthase
dimerization and function in the rat gastric antrum
Pandu R. R. Gangula,1 William L. Maner,2 Maria-Adelaide Micci,1
Robert E. Garfield,2 and Pankaj Jay Pasricha1
1
Division of Gastroenterology and Hepatology, Department of Internal Medicine, and 2Department of Obstetrics
and Gynecology, Reproductive Sciences, The University of Texas Medical Branch, Galveston, Texas
Submitted 31 August 2006; accepted in final form 16 November 2006
Gangula PRR, Maner WL, Micci M-A, Garfield RE, Pasricha
PJ. Diabetes induces sex-dependent changes in neuronal nitric oxide
synthase dimerization and function in the rat gastric antrum. Am J
Physiol Gastrointest Liver Physiol 292: G725–G733, 2007;
doi:10.1152/ajpgi.00406.2006.—Diabetic gastroparesis is a disorder
that predominantly affects women. However, the biological basis of
this sex bias remains completely unknown. In this study we tested the
hypothesis that a component of this effect may be mediated by the
nitrergic inhibitory system of the enteric nervous system. Age-
matched male and female Sprague-Dawley rats were studied 8 or 12
wk after streptozotocin (55 mg/kg body wt ip)-induced sustained
hyperglycemia and compared with controls. Solid gastric emptying
(GE) studies were performed in all the groups. Changes in gastric
antrum neuronal nitric oxide synthase (nNOS) mRNA and protein
levels were analyzed by real-time PCR and Western immunoblotting,
respectively. nNOS dimerization studies were performed using low-
temperature SDS-PAGE. In vitro nitrergic relaxation (area under
curve/mg tissue wt) was studied after the application of electric field
stimulation in an organ bath. Changes in intragastric pressure
(mmHg䡠s) in freely moving rats in the presence or absence of NG-
nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) were
examined by an ambulatory telemetric method. After diabetes induc-
tion, GE is delayed in both male and female rats. However, diabetic
females exhibited significant delayed GE than in diabetic males.
Compared with male controls, gastric nNOS expression and nitrergic
relaxation were substantially elevated in healthy female control rats,
accompanied by significantly reduced intragastric pressure. The active
dimeric form and dimer-to-monomer ratio of nNOS␣ were also higher
in healthy females compared with male rats (P ⬍ 0.05). Diabetic
females, but not males, showed significant (P ⬍ 0.05) impairment in
both gastric nNOS␣ dimerization and nitrergic relaxation, accompa-
nied by an increase in intragastric pressure. Our data provide evidence
that females may have a greater dependency on the nitrergic mecha-
nisms in health. Furthermore, diabetes seems to affect the nitrergic
system to a greater extent in females than in males. Together, these
changes may account for the greater vulnerability of females to
diabetic gastric dysfunction.
solid gastric emptying; nitrergic relaxation; intragastric pressure
GASTRIC DYSMOTILITY OR GASTROPATHY occurs in 20 –55% of
patients with Type 1 (insulin dependent) and up to 30% of
patients with Type 2 diabetes (non-insulin dependent) (37).
Symptoms of diabetic gastropathy can range from mild dys-
pepsia to recurrent vomiting and abdominal pain and are often
associated with delayed or accelerated gastric emptying. Fun-
dic tone abnormalities and/or poor antroduodenal coordination
Address for reprint requests and other correspondence: P. R. R. Gangula,
Univ. of Texas Medical Branch, Dept. of Internal Medicine, Division of
Gastroenterology/Hepatology, 301 University Blvd., Galveston, TX 77555-
0632 (e-mail: pgangula@utmb.edu).
http://www.ajpgi.org 0193-1857/07 $8.00 Copyright © 2007 the American Physiological Society
has been reported in diabetic patients (28). Although the
pathogenesis of diabetic gastropathy is not completely under-
stood, it appears to be much more common (up to four times)
in women (46, 22). Such a sex bias is also seen in the idiopathic
variety of gastric dysfunction, reinforcing the concept of
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women being more susceptible to this condition. It is not clear,
however, whether this phenomenon suggests a unique biolog-
ical vulnerability in females or simply an exaggeration of
preexisting differences in gastric motility. Thus gastric
antroduodenal motility is slower even in healthy women,
perhaps owing to hormonal influence (2). Premenopausal
women, pregnant women during labor, and postmenopausal
women receiving hormone replacement therapy develop re-
versible gastrointestinal complications including delayed gas-
tric emptying for both solids and liquids compared with age-
matched men (3, 4, 27). Moreover, studies using male and
female rats indicate that estradiol-17␤, either alone or in
combination with progesterone, may cause delayed gastric
emptying (16).
Gastric motility is regulated in large part by neurons of the
enteric nervous system located in the muscle wall (53). These
neurons are either excitatory (releasing acetylcholine) or inhib-
itory [releasing nitric oxide (NO) and vasoactive intestinal
peptide]. NO is the principal nonadrenergic noncholinergic
(NANC) inhibitory neurotransmitter in the gastrointestinal
tract and is produced by neuronal NO synthase (nNOS),
expressed in inhibitory enteric neurons (48). NO activates
soluble guanylate cyclase, producing an increase in the intra-
cellular cGMP leading to muscle relaxation (14, 48). Nitrergic
signaling (from NO-releasing neurons) plays a critical role in
the control of gastric accommodation and pyloric relaxation in
response to a meal. The importance of NO in gastric function
was established by the findings of pyloric hypertrophy and
gastric dilation in mice with a targeted genomic deletion of
nNOS (nNOS⫺/⫺ mice) (19, 32). Vagal modulation of enteric
neuronal function (both inhibitory and excitatory) also plays an
important role in gastric physiology and is predominantly
cholinergic in character (29).
Unlike two other NO synthase (NOS) enzymes (endothelial
and inducible NOS), both full-length and NH2-terminally trun-
cated forms of nNOS exist because of alternative splicing of 5⬘
regions of the nNOS gene in both humans and rodents (36, 41,
42). The predominant form of nNOS in the enteric nerves of
the stomach is nNOS␣, which contains a PDZ/GLGF motif
encoded by exon 2 in the NH2-terminal region (41). The
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of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
G725
G726 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION
PDZ/GLGF domain interacts with various proteins and deter-
mines nNOS␣ subcellular localization and function (9, 47). In
contrast, the NH2-terminally truncated nNOS␤ and nNOS␥
lack the PDZ/GLGF motif for protein-protein interaction and
possible membrane association. nNOS⫺/⫺ mice lacking exon 2
and subsequent membrane-associated full-length nNOS␣ ex-
hibit delayed gastric emptying of solids and liquids (19, 32).
The catalytic activity of NOS depends on dimerization of two
NOS polypeptides (25). Dimerization results in the creation of
high-affinity binding sites for (6R)-tetrahydrobiopterin (BH4)
and arginine in the oxygenase domain and enables electron
transfer between flavin and heme groups (7, 17). Enzymatic
uncoupling of NOS due to lack of BH4 may in part account for
reduced NO production and increased oxidative stress factors
such as superoxide (26, 51). The importance of dimerization
for the catalytic activity of endothelial NOS and its disruption
in diabetes has been reported by several independent investi-
gators (13, 33). However, to the best of our knowledge, there
have been no previous in vivo studies on gastrointestinal nNOS
dimerization and function.
Although delayed or accelerated gastric emptying has long
been taken as a hallmark of diabetes, in recent reports most
experts concur that this correlates poorly if at all with clinical
symptoms (37, 38). Nitrergic relaxation in gastric muscle
preparations obtained from diabetic biobreeding/Worcestor
(BB/W; an animal model of human Type 1 diabetes) rats is
significantly impaired, along with a decrease in nNOS-immu-
noreactive cells in the gastric myenteric plexus and mRNA
expression (49). Similar changes have also been shown in
obese and streptozotocin (STZ)-induced diabetic mice and rats
(21, 52). Generally, these changes in phenotypic expression
have not been accompanied by neuronal loss and, indeed, have
been shown to be reversible by insulin treatment (14, 52). It is
noteworthy, however, that these studies have all been performed
in male animals, despite the knowledge that both nitrergic tissue
relaxation and nNOS in gastric fundus can be enhanced by
estrogen treatment in female rats (45). Since irregular antral
motility may be an important factor responsible for altered
gastric emptying in patients, we focused on possible distur-
bances in nitrergic regulation of gastric motility in the diabetic
state (18, 50). The results of our study suggest that nNOS
expression, dimerization, and function are sex dependent and
furthermore that diabetes affects these processes differentially
in males and females.
MATERIALS AND METHODS
Experimental rats and induction of diabetes mellitus with STZ. All
procedures were approved by the Institutional Animal Care and Use
Committee at the University of Texas Medical Branch in accordance
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. Adult male and female nonpregnant rats (7 wk
old) were purchased from Harlan Sprague Dawley (Houston, TX).
After arrival at the animal care facility, all rats were maintained in the
colony room with a 12:12-h fixed light-dark photoperiod. Animals
were allowed free access to water and rodent chow.
Diabetes was induced in groups of male (n ⫽ 10) and female (n ⫽
10) rats with a single STZ injection of 55 mg/kg body wt ip (Sigma
Chemical, St. Louis, MO) prepared in 5 mM citrate buffer (vehicle),
pH 4.0 (30). Control groups were injected with the vehicle only.
Blood glucose levels from overnight fasting animals were obtained
48 h after STZ to confirm that diabetes was induced in treated animals.
Similarly, blood glucose levels were also obtained from overnight
AJP-Gastrointest Liver Physiol • VOL
fasting male and female diabetic as well as control rats at the time the
experiment was performed (12 wk after STZ). Diabetes was con-
firmed if blood glucose levels ranged from 300 – 400 mg/dl in both
male and female rats. No differences in blood glucose levels were
noticed between male and female rats 12 wk after the induction of
diabetes with STZ. Age-matched control male and female rats showed
blood glucose levels between 80 –95 mg/dl. Both control and diabetic
rats were selected during the diestrous stage of the estrous cycle. As
reported earlier, we noticed that 60 –70% of diabetic rats show a
persistent diestrous stage of the estrous cycle (data not shown) (8, 24).
Solid gastric emptying studies. Solid gastric emptying studies were
performed in healthy male and female rats. Similarly, gastric empty-
ing studies were performed in both male and female ras 8 wk after
diabetes induction. The gastric emptying rate of a solid meal was
measured as reported previously (31). Groups of healthy and diabetic
rats from both sexes were fasted for 20 –24 h. A known amount of
solid meal with free access to water was supplied for a 3-h period.
Food and water were then removed, and the gastric emptying rate of
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the ingested meal was assessed 4 h later. Following euthanization, the
abdominal cavity was opened, the pylorus and cardia were clamped,
and the stomach was removed. The amount of food contained in the
stomach and solid food ingested by the animals were calculated. The
rate of gastric emptying during the 4-h experimental time was calcu-
lated according to the following equation: gastric emptying (% in
4 h) ⫽ (1 ⫺ gastric content/food intake) ⫻ 100.
Organ bath studies. For studies on NANC activity, the gastroin-
testinal tract from the lower esophageal sphincter to the distal duo-
denum was removed from the body cavity and placed in Krebs
bicarbonate solution gassed with a mixture of 95% O2 and 5% CO2.
The Krebs (pH 7.4) buffer contains (in mM) 118.0 NaCl, 4.7 KCl,
25.0 NaHCO3, 1.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 11.5 glucose.
Circular gastric antrum muscle strips were mounted between two
L-shaped tissue hooks in 5-ml water-jacketed organ baths containing
Krebs buffer at 37°C and continuously bubbled with 95% O2-5% CO2
(Radnoti Glass Technology, Monrovia, CA). Tension for each muscle
strip was monitored with an isometric force transducer and analyzed
by a digital recording system (Biopac Systems, Santa Barbara, CA).
Tension was increased progressively, and contractile responses to
potassium chloride (60 mM) were analyzed in Krebs bicarbonate
solution measured in various resting conditions (45, 52). Gastric
antrum neuromuscular strips were preincubated for 30 min with
atropine (10 ␮M), phentolamine (10 ␮M), and propranolol (10 ␮M) to
block cholinergic and adrenergic mediated responses. Tone was raised
60 – 80% of maximal contraction produced by 60 mM KCl by addition
of 5-hydroxytryptamine (5-HT) to a final concentration of 100 ␮M.
Specimens demonstrating sustained tonic contractions with 5-HT
stimulation were used for the experiments. Nitrergic relaxations were
induced for 60 s after contraction with 5-HT by electric field stimu-
lation (EFS; 90 V, 2 Hz, 1-ms pulse for a duration of 1 min, as
indicated) (Grass stimulator model SD9, Grass Instruments, Astro-
Med, West Warrick, RI). The NO dependence of nitrergic relaxations
was confirmed by preincubation with NG-nitro-L-arginine methyl ester
(L-NAME, 100 ␮M; 30 min). All drugs used in this study were
purchased from Sigma Chemical. To confirm the role of neuronal
depolarization in evoking NANC relaxations, gastric tissues were
preincubated for 30 min in the presence of TTX (1 ␮M; Calbiochem).
At the end of each experimental protocol, the muscle strip was
removed, blotted dry with filter paper, and weighed.
Comparisons between the groups were performed by measuring the
area under the curve (AUC) of the EFS-induced relaxation (AUCR)
for 1 min and the baseline for 1 min (AUCB) according to the formula
(AUCR ⫺ AUCB)/weight of tissue (mg) ⫽ AUC/mg of tissue (34).
Ambulatory telemetric studies. Gastric contractility in control and
diabetic rats was measured by using an ambulatory telemetric exper-
imental apparatus previously reported for quantifying uterine contrac-
tility (10, 11).
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 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION
Groups (n ⫽ 3– 4) of diabetic and control rats were anesthetized
with ketamine (45 mg/kg body wt; Fort Dodge Laboratory, Fort
Dodge, IA) and xylazine (5 mg/kg body wt; Burns Veterinary Supply,
New York, NY). A pressure telemetry transducer/transmitter (C50-
PXT model, Data Sciences, Arden Hills, MN) was implanted into the
abdomen of each animal. The transducer pressure catheter was intro-
duced into the gastric antrum cavity 5 mm proximal to the pylorus to
record intragastric pressure (IGP, mmHg䡠s). After the baseline IGP
was recorded, L-NAME (200 mg䡠day⫺1䡠kg body wt⫺1 per rat) was
administered subcutaneously in the same rats by osmotic minipumps
(Alza, Palo Alto, CA, model 2ML1 with a pumping rate of 10 ␮l/h)
for 4 days. All subsequent studies on these animals were performed 1
wk after surgery in overnight fasted rats that were awake and in a
free-moving state. Pressure recordings were performed at least 2–3 h
between 9:00 AM and 12:00 noon throughout the studies.
Data were transmitted by telemetry (RLA 1020 telemetry receivers,
Data Sciences), multiplexed (BCM consolidation matrix, Data Sci-
ences), and sent to an adapter where the signal was then demulti-
plexed, sampled at 1,024 Hz, and converted to analog (UA-10 uni-
versal adapter, Data Sciences). This output was band-pass filtered and
amplified. The final sampling rate was 10 Hz (10, 11). The informa-
tion was then fed to a recording system (MacLab 16/s, AD Instru-
ments; Castle Hill, Australia) and stored for later analysis.
For each animal’s data analysis, pressure recordings for at least 10
consecutive contraction events were averaged at each time point. A
contraction event was defined as a rapidly occurring increase in IGP
of 5 mmHg or more, such that the increased IGP persisted for 5 s or
longer (Fig. 1). The pressure integral (or area under the pressure
curve) was found for each animal at each time point investigated.
Real-time RT-PCR. Total tissue RNA was isolated from the rat
gastric antrum neuromuscular tissues by a single-step guanidine
thiocyanate method using the reagent Trizol (BRL, Gaithersburg,
MD). The quality of RNA was determined by NanoDrop (NanoDrop
Technologies), and the quantity was estimated by an Agilent 2100
Fig. 1. Intragastric pressure (IGP) contractions were observed using implanted
telemetric IGP devices. Contractions were defined as rapidly increasing pres-
sure events, 5 mmHg or more in amplitude, with a duration of 5 s or more.
These pressure curves were isolated and an integral from baseline was
calculated. Ten such consecutive contractions were evaluated in this way to
obtain a mean IGP for each animal for each time point. Means and SE of the
IGP were then found for each group of animals at each time point.
AJP-Gastrointest Liver Physiol • VOL
G727
bioanalyzer (Agilent Technologies, Houston, TX). One microgram of
total RNA was denatured at 65°C for 5 min and cDNA synthesis was
then performed at 42°C for 1 h by using Superscript reverse transcrip-
tase (BRL). An aliquot of generated cDNA was amplified with a pair
of primers (accession number NM_052799) for nNOS (forward 2782–
2800, 5⬘-ACGGACCCGACCTCAGAGA3⬘, reverse 2856 –2837, 5⬘-
CGAGGCCGAACACTGAGAAC-3⬘) and probe [2810 –2835, 5⬘-
(FAM)-AAGTACTGGACCCCTGGCCAATGTGA-(TAMARA)].
Quantitative RT-PCR amplification was performed by using two-step
TaqMan Universal PCR master mix and the ABI Prism 7900 sequence
detection system (Applied Biosystems, Foster City, CA). Cycling
conditions were 50°C for 2 min, 95°C for 10 min, followed by 40
repeats of 95°C for 0.15 min and 60°C for 1 min. Relative amounts of
mRNA were normalized by 18S and calculated threshold cycle
numbers (CT), i.e., 2⫺⌬⌬CT, according to the manufacturer’s sugges-
tion (Applied Biosystems). All studies were performed in the Molec-
ular Genomics Core Laboratory, The University of Texas Medical
Branch, Galveston, TX.
Western blot analysis. We examined nNOS protein expression and
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dimerization using COOH- and NH2-terminal antibodies, respec-
tively. COOH-terminal antibody detects all (total) forms of nNOS (␣,
␤, and ␥) whereas NH2-terminal antibody detects only the full-length
nNOS␣ protein (41). Under normal conditions, both dimers and
monomers of nNOS protein are intensified at 155 kDa. However,
low-temperature SDS-PAGE of nonboiled sample homogenates sep-
arates NOS dimers (310 kDa) and monomers (155 kDa) (23).
Equal amounts of total protein (40 ␮g each; Pierce Kit, Rockford,
IL) from each preparation were resolved on a 6% SDS-polyacryl-
amide gel, transferred to a nitrocellulose membrane, and primed with
primary COOH-terminal nNOS monoclonal antibody (1:1,000, Trans-
duction Laboratory, San Jose, CA) overnight at 4°C (52). To inves-
tigate nNOS homodimer formation in gastric antrum tissues, low-
temperature SDS-PAGE was employed with nonboiled samples by
using specific NH2-terminal (1–195 amino acids of nNOS␣, exon 2
region) nNOS polyclonal antibody (1:200, Zymed) as previously
described (23). The membrane was washed three times with 20
mmol/l Tris䡠HCl, pH 7.6, 0.05% Tween 20, 100 mM NaCl and then
incubated with a respective secondary antibody, goat anti-mouse and
anti-rabbit coupled to horseradish peroxidase (Amersham Life Sci-
ences, Arlington Heights, IL). After three washes, the membrane was
developed by using the enhanced chemiluminescence system (ECL,
Amersham Pharmacia Biotech, Piscataway, NJ) according to the
manufacturer’s protocol. To verify equal loading of sample protein,
the immunoblots were stripped in stripping solution (Pierce, Rock-
ford, IL) and reprobed with monoclonal anti-␥-tubulin antibody (1:
10,000; Sigma Chemical). Densitometry analysis was performed in
the linear range using a Fluorchem Analysis System (Alpha InfoTech;
San Leandro, CA) and the amount of total nNOS (155 kDa) was
normalized to the ␥-tubulin (48 kDa) signal.
Statistical analysis. Results are expressed as means ⫾ SE obtained
from four to seven animals for solid gastric emptying and organ bath
studies and three to four samples for both IGP and nNOS expression
studies. Data were analyzed for statistical differences with Student’s
t-test or two-way ANOVA followed by the Bonferroni t-test to verify
the differences between individual groups. P ⬍ 0.05 was considered
significant (n ⫽ 3– 6).
RESULTS
Sex-dependent gastric emptying rate in control and diabetic
rats. Figure 2 shows differences in solid gastric emptying rate
in healthy and diabetic male and female rats. Healthy females
during the diestrous stage of their estrous cycle (when circu-
latory estrogens are beginning to be elevated) exhibited a trend
toward a delay in gastric emptying for solids compared with
age-matched male rats. However, this was not statistically
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G728 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION

male controls (Fig. 4C), an effect that was at least partly

reversed by L-NAME administration (Fig. 4C), suggesting
relative preservation of nitrergic tone.
Sex-dependent nNOS expression in gastric tissues in control
and diabetic rats. Compared with their male counterparts,
female controls had higher nNOS mRNA (Fig. 5A), but not
total protein levels (Fig. 5B). Diabetes did not appear to affect
the expression of either mRNA or protein in females. However,
both mRNA and total protein expression was significantly
(P ⬍ 0.05) elevated in gastric antrum obtained from male
diabetic rats compared with their respective controls (Fig. 5).
Dimerization of nNOS␣ in gastric antrum. We next set out
to determine changes in dimerization of nNOS␣ in the various
experimental states. We examined this by low-temperature
SDS-PAGE so that the SDS-resistant dimeric form of nNOS␣
could be measured. This assay is not a measure of the absolute
dimeric content under native conditions, but it is a measure of

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the amount of stable dimer that is not dissociated by SDS and
thus somewhat underestimates the total dimeric content. None-
theless, it is a convenient and reliable measure for studying
Fig. 2. Sex-dependent changes in solid gastric emptying in healthy and
relative changes in the dimeric state of nNOS. We found that
diabetic rats. Diabetes was induced with a single injection of streptozotocin the ratio of nNOS␣ dimers to monomers was significantly
(STZ; 55 mg/kg body wt ip), and all studies were performed after 8 wk of increased (P ⬍ 0.05) in female control rats compared with
diabetes induction. The control group received vehicle (citrate buffer). Fasted male controls in antrum (Fig. 6). Diabetes resulted in signifi-
rats were exposed to normal rat chow ad libitum for a 3-h period and gastric cant (P ⬍ 0.05) decreases in nNOS␣ dimerization in female
emptying was monitored 4 h later. Data are means ⫾ SE of 4 –7 animals per
group. *P ⬍ 0.05 (ANOVA). antral tissue but not in males, compared with their respective
controls (Fig. 6).
Figure 7 shows the changes in gastric emptying (A), gastric
significant. Eight weeks after diabetic induction, a significant antrum nitrergic relaxation (B) and nNOS␣ protein dimer
delay in gastric emptying rate was noticed in both male and
female rats, compared with their respective controls (Fig. 2).
Furthermore, gastric emptying was reduced to a significantly
greater extent in diabetic females compared with diabetic
males.
Sex-dependent nitrergic relaxation in control and diabetic
rats. Circular muscle strips from gastric antrum from both
control and diabetic rats showed nitrergic relaxation in re-
sponse to EFS using a frequency of 2 Hz. This relaxation was
significantly antagonized by preincubation with the NOS in-
hibitor L-NAME (100 ␮M) and completely abolished in the
presence of TTX (data not shown), confirming its nitrergic
nature and neuronal source, respectively (Fig. 3).
Tissues obtained from female control showed substantially
greater nitrergic relaxation compared with the male control
group (P ⬍ 0.05). Furthermore, nitrergic relaxation was sig-
nificantly impaired (P ⬍ 0.05) in female diabetic rats but not in
male diabetics, compared with their respective control groups
(Fig. 3).
Sex-dependent IGP in control and diabetic rats. Intraluminal
antral pressure was measured in age-matched male and female
controls (Fig. 4A) and 12 wk after induction of diabetes in
controls and in L-NAME-treated animals (Fig. 4B). As shown
in Figs. 4C and 3D, the female control group showed a Fig. 3. Effect of diabetes on gastric antrum nonadrenergic and noncholinergic
significant (P ⬍ 0.05) reduction in IGP (mmHg䡠s) compared (NANC) relaxation in response to transmural nerve stimulation (90 V, 2 Hz)
in age-matched male and female rats. Gastric antrum muscle tissues obtained
with male controls. L-NAME infusion attenuated the lowered from 12-wk-old rats were preincubated with atropine, phentolamine, and
antral pressure in female controls, implicating a role for the propranolol (10 ␮M each) and active tone was induced initially with 100 ␮M
nitrergic system in this phenomenon. Diabetes caused a signif- 5-hydroxytryptamine. The nitric oxide (NO) dependence of the NANC relax-
icant (P ⬍ 0.05) IGP elevation in female diabetic rats com- ations in control and diabetic groups was confirmed by preincubation with the
NO inhibitor nitro-L-arginine methyl ester (L-NAME; 100 ␮M). Each point
pared with female controls (Fig. 4D), and this did not change represents means ⫾ SE from 4 – 6 animals in each group. *Significant inhibi-
after L-NAME infusion, suggesting loss of nitrergic tone. By tion with L-NAME; #P ⬍ 0.05 for female control compared with male control;
contrast, IGP was reduced in male diabetic rats compared with $P ⬍ 0.05 for female diabetic compared with female control.

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Fig. 4. A: comparison of IGP (mmHg䡠s) for male control vs. female control animals. We noticed that the male animals tend to have more powerful contractions
in general, but roughly the same number of contractions per unit time. In fact, the integral of this activity is significantly higher for the male controls compared
with female controls. B: comparison of control vs. nitric oxide synthase inhibitor, L-NAME-treated (200 mg/kg body wt sc per rat for 4 days) female animals.
Notice that the L-NAME-treated animals tend to have generally more powerful and more frequent contractions. In fact, the integral of this activity is significantly
higher for the L-NAME-treated animals compared with female controls. C and D: effect of diabetes on IGP in age-matched male (C) and female (D) rats. IGP
were measured using ambulatory telemetric devices (see MATERIALS AND METHODS) in the presence or absence of L-NAME (200 mg/kg body wt sc per rat for
4 days) administration by osmotic minipumps in conscious, unrestrained animals. Each point represents means ⫾ SE from 3– 4 animals in each group. *P ⬍
0.05.

levels (C) in diabetic male and diabetic females after normal-
ization to their own controls. As can be seen, diabetes signif-
icantly (P ⬍ 0.05) reduced gastric emptying, nitrergic relax-
ation, and nNOS␣ protein dimer levels in females compared
with male rats (Fig. 7). The decline in gastric antrum nNOS␣
dimer levels correlated with changes in gastric antrum nitrergic
relaxation and solid gastric emptying in diabetic female rats
(Fig. 7).
DISCUSSION
Abnormal gastrointestinal motility in diabetes mellitus is
likely multifactorial in origin, reflecting disturbances in enteric
and vagal neural activity as well as interstitial cells of Cajal and
smooth muscle function (20, 28, 33, 38). Of these, enteric
neuropathy may be particularly important (5, 6, 48). Several
studies of animal models of diabetes have convincingly shown
disturbances in enteric nerves, particularly involving nitrergic
nerves (14, 49, 52). Since these studies have used male rats
exclusively, it has been difficult to correlate these findings with
the clinical finding that 80% of all gastroparetic patients are
female. It is possible that the pathogenetic mechanisms of
AJP-Gastrointest Liver Physiol • VOL
diabetic gastric dysmotility are common to both men and
women but the latter are disproportionately symptomatic be-
cause the motility of their stomachs is slower to begin with. On
the other hand, it is also possible that there are fundamental
sex-specific differences between how the gastric neuromuscu-
lar apparatus is affected by diabetes. The results of our study
suggest that the latter explanation may be more likely.
In this study, we have demonstrated sex differences in solid
gastric emptying, antral nNOS expression, nitrergic relaxation,
IGP, and nNOS␣ protein dimerization in both diabetic and
nondiabetic rats. Compared with control male rats, healthy
females during the diestrous stage of the estrous cycle showed
delayed gastric emptying (Fig. 2). However, this was not
statistically significant. Previous studies have demonstrated
that estradiol-17␤ administration delays gastric emptying for
liquids in both male and female rats (16). Thus, more studies
are warranted to address whether solid gastric emptying rate is
different during various stages of the estrous cycle when
estrogen levels are higher than in the diestrous phase. How-
ever, nitrergic relaxation of the antrum is clearly greater in
healthy diestrous females (Fig. 3) compared with males, and
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G730 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION

this correlates with a corresponding increase in both total

nNOS protein expression (Fig. 5) and nNOS␣ dimerization
(Fig. 6). Our studies further demonstrate that IGP is signifi-
cantly lower in females compared with male rats (Fig. 4, C and
D). This is in keeping with human studies showing that gastric
antral contractility and gastric emptying is slower in young
women compared with men whereas circulatory estrogen lev-
els are elevated (27). Their method for measuring antral con-
tractility was not telemetric, but since the study was performed
in humans, the externalized catheters posed little problem for

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Fig. 6. Sex- and diabetes-related changes in the expression of neuronal nitric
oxide-␣ dimerization (nNOS␣) in the gastric distal antrum of male control,
female control, male diabetic, and female diabetic rats. Equal amounts of
protein (40 ␮g) were loaded on each lane and detected with polyclonal
antibody specific for the exon 2 encoded NH2-terminal domain of nNOS␣.
Top: representative immunoblot of gastric distal antrum homogenates (n ⫽
3– 4) showing the ratio of nNOS␣ dimers (310 kDa) to monomers (155 kDa).
Bottom: densitometric analysis followed by a ratio of nNOS␣ dimerization to
␥-tubulin is calculated. Bars represent means ⫾ SE. Significant differences
from control vs. diabetes are noted. #P ⬍ 0.05 for female control compared
with male control and $P ⬍ 0.05 for female diabetic compared with female
control.

implementation insofar as the integrity of the data is con-

cerned. In animals this is not the case, where with externalized
catheters, either the animal must be restrained (introducing
undue stress) to prevent destruction of the measurement appa-
ratus or the data must be acquired while the animals are

Fig. 5. Sex- and diabetes-related changes in the expression of neuronal nitric

oxide synthase (nNOS) in the gastric distal antrum of male control, female
control, male diabetic, and female diabetic rats. nNOS mRNA (A) and protein
(B) levels were measured from gastric antrum by quantitative RT-PCR and
Western blot analysis, respectively. Diabetes was induced with a single
injection of STZ (55 mg/kg body wt ip) and all studies were performed after
12 wk of diabetes induction. The control group received vehicle (citrate
buffer). A: nNOS and 18S mRNA expressions were measured by real-time
RT-PCR. Relative abundance of nNOS mRNA normalized by 18S was
calculated from threshold cycles (CT), i.e., 2⫺⌬⌬CT. B: representative immu-
noblot analysis of gastric distal antrum homogenates shows the expression of
nNOS (155 kDa) and ␥-tubulin (48 kDa) (top). Equal amounts of protein (40
␮g) were loaded on each lane and detected with monoclonal antibody specific
for the COOH-terminal domain of nNOS. Densitometric analysis followed by
ratio of nNOS to ␥-tubulin are calculated (bottom). Bars represent means ⫾ SE
(n ⫽ 3– 4). Significant differences from control vs. diabetes are noted. *P ⬍
0.05 for male diabetic compared with male control; #P ⬍ 0.05 for female
control compared with male control.

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 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION G731

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Fig. 7. Sex-dependent changes in solid gastric emptying (A), gastric antrum nitrergic relaxation (B) and gastric antrum nNOS dimer levels (C) in healthy and
diabetic rats. Diabetes was induced with a single injection of STZ (55 mg/kg body wt ip). The control group received vehicle (citrate buffer). Diabetes reduced
solid gastric emptying (A), nitrergic relaxation (B), and nNOS␣ protein dimer levels (C) in diabetic female gastric tissues compared with diabetic male rats. The
changes in the above parameters were normalized with their respective control group. Data were presented as % change over control. *P ⬍ 0.05 (ANOVA).

unconscious. Both of these alternatives may lead to spurious
data. By using a telemetric device in conscious, unrestrained
animals, we obtained the most accurate measurement for in-
traluminal pressure. In our study, L-NAME infusion increased
IGP significantly in female but not in male rats, suggesting that
nonnitrergic mechanisms may be more important in the latter
(Fig. 4, C and D). Thus it appears that females rely on nitrergic
control of gastric motility to a greater extent than males and
hence may be more vulnerable to alterations of this system
induced by diabetes.
We next turned our attention to changes in these parameters
after induction of diabetes. The rate of gastric emptying may
AJP-Gastrointest Liver Physiol • VOL
vary depending upon the type of method employed, i.e., liquids
vs. solids; type, duration, and severity of diabetes. Human
studies have demonstrated that diabetic women have more
delayed gastric emptying compared with diabetic men (22, 46).
In the present study, we have demonstrated that solid gastric
emptying rate is significantly slower in both male and female
rats after diabetic induction compared with their respective
controls. Furthermore, these studies suggested that diabetic
females showed significant delayed gastric emptying compared
with diabetic males. These studies together with clinical find-
ings suggested that gastric emptying is slower in diabetic
females than in diabetic males.
292 • MARCH 2007 • www.ajpgi.org
G732 DIABETES IMPAIRED GASTRIC NNOS DIMER FUNCTION
In diabetic gastric dysfunction, antral motility and the coor-
dination of pressures between the antrum and duodenum are
diminished (28, 46, 53). Antral hypomotility has been recorded
with intraluminal pressure transducers in patients with diabetes
mellitus. In this study, we have shown that nitrergic relaxation
(Fig. 3) is significantly reduced with concomitant increases in
IGP in female diabetic rats (Fig. 4D). Furthermore, nNOS␣
dimerization (Fig. 6) but not total (Fig. 5) nNOS expression is
significantly reduced in these animals. After normalization
with their own control group, diabetic females exhibited sig-
nificant reduction in solid gastric emptying (Fig. 7A), gastric
antrum nitrergic relaxation (Fig. 7B), and nNOS␣ protein
dimer levels (Fig. 7C) compared with male rats. These data
suggest that a decline in nNOS␣ dimerization correlates with
changes in nitrergic relaxation and gastric emptying and may
explain why females have a greater tendency to gastroparesis.
In this study, we also demonstrate an increase in gastric
antrum nNOS expression in the male diabetic group. Although
our results are similar to those reported by Adeghate et al. (1),
they are in contrast to most of the previous literature (all of
which deals exclusively with male rats) reporting a decrease in
gastric nNOS expression in diabetic rats (48, 49, 52). We are
unable to satisfactorily explain this discrepancy but do note
that we have utilized a lower dose of STZ to induce diabetes
compared with others (15, 49). Furthermore, by contrast to
female diabetic rats, we were unable to show a significant
reduction in nNOS␣ dimerization or nitrergic relaxation in
male diabetic animals. Interestingly, diabetes resulted in re-
duced IGP in males (compared with an increase in females),
again suggesting a potentially important sex difference in the
control of gastric motility (Fig. 4, C and D). The mechanism
underlying the reduction in IGP in diabetic males remains
unknown but may be due to other factors such as impairment
of interstitial cells of Cajal dysfunction and antral electrical
pacemaking and neurotransmitter coupling. More studies are
needed to understand the biological basis of this sex effect.
The mechanisms regulating changes in nNOS dimerization
and nitrergic function in female diabetic animals are not
addressed in the present investigation but may be related to
alterations in female hormonal control. Circulatory estradiol
levels are lower in diabetic women and rats (30, 39). Estradiol-
17␤ increases both nNOS expression and GTP cyclohydrolase
1 expression (43, 44). The latter is a rate-limiting enzyme for
the production of BH4, which is required for dimerization of
nNOS (12). Therefore, we suggest that the decrease in estradiol
in diabetic female gastric tissues results in a reduced BH4
synthesis with adverse effects on nNOS activity, thus leading
to increased gastric dysmotility. Studies are in progress to
address this issue.
In summary, this study demonstrates significant sex differ-
ences in gastric emptying, gastric antral nitrergic function, and
nNOS expression and dimerization in both health and disease.
Nitrergic relaxation is more pronounced in healthy females,
accounted for, perhaps, by an increased expression of the
active dimeric form of nNOS␣. On the other hand, chronic
hyperglycemia causes a greater reduction in active forms of
nNOS in females associated with significantly greater impair-
ment of nitrergic relaxation. This study also illustrates for the
first time the importance of nNOS␣ dimerization in gastric
physiology. These findings may provide a biological explana-
AJP-Gastrointest Liver Physiol • VOL
tion for the greater vulnerability of females to develop diabetic
gastric dysmotility problems.
ACKNOWLEDGMENTS
The authors thank Xiemin Cao for technical assistance and John Winston
and Raj Kumar for thoughtful discussions. Also, we thank Dr. Shao-Qing Shi
for help with the surgeries for telemetric studies.
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